A mass spectrometry detection method based on single-base sequence mass label and its application

Abstract

The present invention discloses a mass spectrometry detection method and application based on a single-base sequence mass label, which utilizes a single-base sequence DNA strand as a mass label for mass spectrometry immunoassay. After the nucleic acid sequence is labeled on the target, the DNA nucleic acid strand is enzymatically hydrolyzed into a single base by exonuclease and alkaline phosphatase, and the concentration of the base after enzymatic hydrolysis is detected by a liquid chromatography-mass spectrometry method to achieve quantification of the target. Compared with the prior art, the mass spectrometry immunoassay signal amplification technology of the present invention is simple, and the higher labeling efficiency makes the method have higher sensitivity, and can be widely used in the fields of biomarker detection and hazardous substance screening. The detection limit of the method of the present invention is much lower than the limit specified in the national standard, and can be used for the detection of low-concentration targets, providing a new method path for the screening of hazardous substances.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a mass spectrometry detection method and application based on single-base sequence mass labels. Background Art

[0002] Immunoassay technology based on mass labeling has been widely used in qualitative and quantitative detection of hazardous factors, drug screening, and tumor marker screening. Mass labels are generally a series of molecules or ions that are easily ionized and can output their characteristic mass-to-charge ratio information. By mass encoding the target to be tested, the concentration or activity of the target molecule can be quantitatively analyzed based on the intensity of its characteristic ion signal. At present, metal elements, organic small molecules, polymers, nucleic acids, peptides, etc. can be used as mass labels for marking targets to be tested.

[0003] CN117191933A discloses a preparation method and application of a mass spectrometry label PTACA, which describes a preparation method of an organic small molecule mass spectrometry label, using a three-step organic reaction to obtain a mass spectrometry label PTACA, wherein the reaction involves a large number of purification steps and hydrogen pressure reactions, which are cumbersome and have a high threshold, and uses organic reagents such as THF, which is not in line with the general trend of green environmental protection. Moreover, the simple mass spectrometry labeling target lacks an amplification strategy, which limits the detection limit of the method.

[0004] CN116751844A discloses a nucleic acid detection kit based on mass coding and its application, which describes a method based on mass coding of single-stranded nucleic acid fragments of specific length, and develops a nucleic acid detection kit based on mass label, which uses single-stranded nucleic acid fragments of specific length as mass labels, and after the double-stranded hybrid formed with the complementary chain is recognized and degraded by the double-stranded specific nuclease, the target nucleic acid can be released and single-stranded nucleic acid fragments of different lengths can be generated, and the mass of single-stranded nucleic acid fragments of different lengths can be detected by mass spectrometry to achieve the effect of detecting the target. However, the detection sensitivity of this method is low, and it needs to go through steps such as temperature change and amplification, which is relatively cumbersome.

[0005] In summary, the existing mass labeling technology has problems such as complicated and cumbersome labeling steps and lack of signal amplification strategy during the labeling process, resulting in insufficient sensitivity of the detection method based on this principle. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a mass spectrometry detection method and application based on a single-base sequence mass label, which utilizes a single-base sequence DNA single strand as a mass label for mass spectrometry immunoassay. After the nucleic acid sequence is labeled on the target, the DNA nucleic acid chain is enzymatically hydrolyzed into a single base by exonuclease and alkaline phosphatase, and the concentration of the base after enzymatic hydrolysis is detected by liquid chromatography-mass spectrometry to achieve quantification of the target. Compared with the prior art, the mass spectrometry immunoassay signal amplification technology of the present invention is simple, and the higher labeling efficiency makes the method have higher sensitivity, and can be widely used in the fields of biomarker detection, hazard screening and the like.

[0007] The present invention is achieved through the following technical solutions:

[0008] A mass spectrometry detection method based on a single base sequence mass tag comprises the following steps:

[0009] Step 1) Preparation of biotin-modified coated antigen: add the target antigen solution to an ultrafiltration tube, centrifuge at 4°C, 10,000 rpm for 5 minutes, discard the filtrate, and repeat 3 times; collect the residual liquid in the ultrafiltration tube; dissolve biotin in DMSO solvent, add the biotin solution to the antigen solution according to the molar ratio of biotin to antigen molecule of 20:1, and react at room temperature for 30 minutes; after the reaction, dialyze the solution to remove free biotin and other reagents; use an ultrafiltration tube to concentrate the biotinylated labeled antigen solution, add an equal volume of glycerol, and store at -20°C;

[0010] Step 2) preparing coated antigen-modified magnetic beads: taking streptavidin-modified magnetic beads in PBS buffer, fixing the magnetic beads by magnetic adsorption, and washing the magnetic beads; adding PBS buffer and the biotinylated labeled antigen solution prepared in step 1), shaking and reacting at 37° C. for 1 hour, then washing with PBST solution to remove unbound biotinylated labeled antigen, and finally storing in the reaction solution;

[0011] Step 3) Preparation of DNA chain modified antibody: Take a single base sequence DNA chain and add the detection target antibody, react in a carbonate buffer solution for 12 hours, dialyze the solution to remove free DNA chains that have not reacted with the antibody; use an ultrafiltration tube to concentrate the DNA chain modified antibody solution, add an equal volume of glycerol, and store at -20°C;

[0012] Step 4) Target detection: Take the DNA chain modified antibody prepared in step 3) and mix it with the test solution at room temperature for 30 minutes, then add the coated antigen modified magnetic beads prepared in step 2), mix at room temperature for 30 minutes, add DNA exonuclease Exonuclease I and alkaline phosphatase, react in the buffer solution matching the exonuclease at 37°C for 1 hour, extract twice, collect the aqueous phase and detect it using a liquid chromatography-mass spectrometer.

[0013] Preferably, in step 1), the concentration of the detection target coated antigen solution is 1 mg / mL; the concentration of the antigen solution is 1 mg / mL; and the final concentration of the biotin solution is 2 mg / mL.

[0014] Preferably, the concentration of the streptavidin-modified magnetic beads in step 2) is 10 mg / mL

[0015] Preferably, the length of the DNA chain of the single base sequence in step 3) is 30 to 100 bp, and the 5' end thereof is modified with an aldehyde group.

[0016] Preferably, in step 3), the concentration of the single-base sequence DNA chain is 100 μM; the concentration of the detection target antibody is 1 mg / mL; the concentration of the carbonate buffer solution is 0.01 M, and the pH is 8.5.

[0017] Preferably, the concentration of the biotinylated labeled antigen solution prepared in step 1) is 2 mg / mL; the concentration of the DNA chain modified antibody prepared in step 3) is 2 mg / mL.

[0018] The application of the above-mentioned mass spectrometry detection method based on single-base sequence mass tags in the screening of hazardous substances.

[0019] Preferably, the hazardous substance is vomitoxin.

[0020] The above-mentioned mass spectrometry detection method based on single-base sequence mass tags is applied in the detection of low-concentration targets.

[0021] Preferably, the detection limit of the low-concentration target detection is <0.01 ng / mL.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) Compared with CN117191933A, the synthesis of mass spectrometry labels in the present invention is relatively simple. The present invention uses bases after enzymatic hydrolysis of single-sequence DNA as mass spectrometry markers, which has the advantage of simple labeling. The existing DNA single-strand synthesis and functionalization technology is mature, and many commercial companies at home and abroad have similar services, which has obvious advantages over traditional organic molecules.

[0024] (2) Compared with CN116751844A, the present invention has high detection efficiency. CN116751844A uses single-stranded DNA as a mass label, while the present invention uses the base after enzymatic hydrolysis as a mass label, and introduces an exonuclease to exocut the DNA into bases, essentially converting the signal of the single-stranded DNA into a signal of multiple bases, thereby achieving signal amplification of the target and improving the sensitivity of detection. Since a single-sequence DNA chain contains a large number of identical bases, detection after enzymatic hydrolysis is equivalent to signal amplification of the target, so the method of the present invention reduces the detection limit compared to simple molecular labeling, and is different from general nucleic acid signal amplification, which requires temperature change, amplification and other steps. The method of the present invention completes the signal amplification step at the DNA synthesis stage, thereby improving detection efficiency and reducing detection time.

[0025] (3) The detection limit of the method of the present invention is lower than 0.01 ng / mL, which is much lower than the limit specified in the national standard "GB 2761-2017 National Food Safety Standard Limits of Mycotoxins in Foods", and can be used for the detection of low-concentration targets, providing a new method for the screening of hazards. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart of the mass spectrometry detection method based on single-base sequence mass tags in Example 1;

[0027] Figure 2 is the chromatogram detected by liquid chromatography-mass spectrometry in Example 1;

[0028] Figure 3 The mass spectrometer peak diagram of liquid chromatography-mass spectrometry detection in Example 1;

[0029] Figure 4 This is the detection result of vomitoxin in Example 1. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1

[0034] The DNA chain sequence used in the method of the present invention is a single base sequence (A, T, C, G), with a length of 30 to 100 bp, modified with an aldehyde group at the 5' end. The nucleic acid fragment in this embodiment was purchased from Sangon Biotech (Shanghai) Co., Ltd., with a sequence of a single base T and an aldehyde group modified at the 5' end, and a length of 100 bp. The streptavidin-modified magnetic beads involved in the experiment were purchased from Wuxi Biomag. Taking vomitoxin as an example of the detection target, vomitoxin antibodies, BSA-modified coated antigens, and alkaline phosphatase were purchased from Biotech (Shanghai) Co., Ltd. Exonuclease I enzyme and matching buffer were purchased from themofisher company.

[0035] A mass spectrometry detection method based on single base sequence mass tags, such as Figure 1 As shown, the specific steps are as follows:

[0036] (1) Preparation of biotin-modified coated antigen

[0037] Add 200 μL of 1 mg / mL vomitoxin-coated antigen solution to an ultrafiltration tube, centrifuge at 4°C, 10,000 rpm for 5 min, discard the filtrate, repeat 3 times, remove possible additives such as sodium azide, and prevent interference with the labeling reaction. Collect the residual liquid in the ultrafiltration tube and adjust the concentration to 1 mg / mL with 0.01M PBS solution. Dissolve biotin in DMSO solvent to a final concentration of 2 mg / mL. Add biotin to the antigen solution at a ratio of 20:1 between biotin and antigen molecules, and react at room temperature for 30 min. After the reaction, place the solution in a dialysis bag (molecular weight 50,000) and dialyze it in 0.01M PBS solution to remove free biotin and other reagents. Then transfer the solution in the dialysis bag to an ultrafiltration tube (molecular weight 50,000), and use the ultrafiltration tube to concentrate the biotinylated labeled antigen solution to adjust the antigen concentration to 2 mg / mL. Add an equal volume of glycerol, and store the solution at -20°C if not used immediately.

[0038] (2) Preparation of antigen-modified magnetic beads

[0039] Take 25 μL of streptavidin magnetic beads (10 mg / mL) in 1 mL of 0.01 M PBS buffer, use magnetic force to fix the magnetic beads, pour out the liquid, and remove the preservatives and other interferences in the magnetic bead storage solution. Add 1 mL of 0.01 M PBS buffer and 5 μL of the biotinylated labeled antigen solution prepared in step (1), shake at 37 ° C for 1 hour, then wash with 0.01 M PBST solution to remove unbound biotinylated labeled antigen, and finally store the magnetic beads in 0.01 M PBS solution.

[0040] (3) Preparation of DNA chain-modified antibodies

[0041] Take 5 μL of 100 μM single-base sequence DNA chain and add 10 μL of 1 mg / mL vomitoxin antibody. React in 0.01 M carbonate buffer solution for 12 hours. Then put the solution into a dialysis bag (molecular weight 30,000) and dialyze against PBS to remove free DNA chains that have not reacted with the antibody. Use an ultrafiltration tube to concentrate the DNA chain modified antibody solution and adjust the concentration to 2 mg / mL. Add an equal volume of glycerol and store at -20°C.

[0042] (4) Target Detection

[0043] Take 5 μL of the DNA chain modified antibody prepared in step (3) and mix it with the test solution at room temperature for 30 minutes, then add 5 μL of the coated antigen modified magnetic beads prepared in step (2), mix at room temperature for 30 minutes, add 100U DNA exonuclease Exonuclease I and 100U alkaline phosphatase, react in the buffer solution matching the exonuclease at 37°C for 1 hour, extract the solution twice with equal volumes of phenol solution and phenol chloroform solution (volume ratio 1:1), collect the aqueous phase and detect it using a liquid chromatography-mass spectrometer.

[0044] (5) Liquid chromatography-mass spectrometry detection

[0045] The chromatographic conditions were as follows:

[0046] Chromatographic column: ACQUITY UPLC BEH C18 (2.1mm×100mm, 1.7μm); mobile phase: A is an aqueous solution (containing 0.1% formic acid, 5mmol / L ammonium formate), B is methanol; gradient program: 0-4min, 5% B; 4-8min, 5%-90% B; 8-10min, 90% B; 10-10.5min, 90%-5% B; 10.5-15min, 5% B; injection volume: 5μL; flow rate: 0.2mL / min; column temperature: 40℃.

[0047] The mass spectrometry conditions were as follows:

[0048] Ion source: jet stream electrospray ion source (AJS ESI), scan mode: positive ion mode, detection mode: multiple reaction monitoring (MRM); nebulizer pressure: 45 psi; gas temperature: 300°C, gas flow rate: 10 L / min; sheath gas (N2) temperature: 350°C, sheath gas (N2) flow rate: 11 L / min; capillary voltage: 3500 V; nozzle voltage: 500 V. Detailed mass spectrometry parameters are shown in Table 1.

[0049] Table 1 Mass spectrometry parameters

[0050]

[0051]

[0052] like Figure 2 As shown, under the above chromatographic conditions, the T base after enzymatic hydrolysis appears on the chromatogram in about 2 minutes and has a good signal intensity.

[0053] like Figure 3 As shown in the figure, under the above mass spectrometry conditions, the mass spectrometry characteristic peaks of the T base after enzymatic hydrolysis are 54.1, 84.0, and 109.9.

[0054] like Figure 4 As shown, when the method of this example is used to analyze vomitoxin in wheat, it shows good linearity in the range of 0.01 to 100 ng / mL, and the linear correlation coefficient is greater than 0.999.

[0055] From the experimental results of this example, it can be seen that the method of the present invention has a sensitive detection effect for small molecule hazards such as vomitoxin, and the detection limit is lower than 0.01 ng / mL, which is much lower than the limit specified in the national standard "GB 2761-2017 National Food Safety Standard Limits of Mycotoxins in Food", indicating that this method has good application value.

[0056] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A mass spectrometry detection method based on single-base sequence mass tags, characterized in that: The following steps are involved: Step 1) Preparation of biotin-modified coated antigen: add the target antigen solution to an ultrafiltration tube, centrifuge at 4°C, 10,000 rpm for 5 minutes, discard the filtrate, and repeat 3 times; collect the residual liquid in the ultrafiltration tube; dissolve biotin in DMSO solvent, add the biotin solution to the antigen solution according to the molar ratio of biotin to antigen molecule of 20:1, and react at room temperature for 30 minutes; after the reaction, dialyze the solution to remove free biotin and other reagents; use an ultrafiltration tube to concentrate the biotinylated labeled antigen solution, add an equal volume of glycerol, and store at -20°C; Step 2) preparing coated antigen-modified magnetic beads: taking streptavidin-modified magnetic beads in PBS buffer, fixing the magnetic beads by magnetic adsorption, and washing the magnetic beads; adding PBS buffer and the biotinylated labeled antigen solution prepared in step 1), shaking and reacting at 37° C. for 1 hour, then washing with PBST solution to remove unbound biotinylated labeled antigen, and finally storing in the reaction solution; Step 3) Preparation of DNA chain modified antibody: Take a single base sequence DNA chain and add the detection target antibody, react in a carbonate buffer solution for 12 hours, dialyze the solution to remove free DNA chains that have not reacted with the antibody; use an ultrafiltration tube to concentrate the DNA chain modified antibody solution, add an equal volume of glycerol, and store at -20°C; Step 4) Target detection: Take the DNA chain modified antibody prepared in step 3) and mix it with the test solution at room temperature for 30 minutes, then add the coated antigen modified magnetic beads prepared in step 2), mix at room temperature for 30 minutes, add DNA exonuclease Exonuclease I and alkaline phosphatase, react in the buffer solution matching the exonuclease at 37°C for 1 hour, extract twice, collect the aqueous phase and detect it using a liquid chromatography-mass spectrometer.

2. A mass spectrometry detection method based on single base sequence mass tags according to claim 1, characterized in that: Step 1) The concentration of the detection target coated antigen solution is 1 mg / mL; the concentration of the antigen solution is 1 mg / mL; the final concentration of the biotin solution is 2 mg / mL.

3. The mass spectrometry detection method based on single base sequence mass tags according to claim 1, characterized in that: Step 2) The concentration of the streptavidin-modified magnetic beads is 10 mg / mL.

4. The mass spectrometry detection method based on single base sequence mass tags according to claim 1, characterized in that: Step 3) The length of the DNA chain of the single base sequence is 30 to 100 bp, and the 5' end thereof is modified with an aldehyde group.

5. The mass spectrometry detection method based on single base sequence mass tags according to claim 1, characterized in that: Step 3) The concentration of the single-base sequence DNA chain is 100 μM; the concentration of the detection target antibody is 1 mg / mL; the concentration of the carbonate buffer solution is 0.01 M, and the pH is 8.

5.

6. The mass spectrometry detection method based on single base sequence mass tags according to claim 1, characterized in that: The concentration of the biotinylated labeled antigen solution prepared in step 1) is 2 mg / mL; the concentration of the DNA chain modified antibody prepared in step 3) is 2 mg / mL.

7. Use of a mass spectrometry detection method based on a single-base sequence mass label according to any one of claims 1 to 6 in screening for hazardous substances.

8. The use according to claim 7, characterized in that: The hazard is vomitoxin.

9. Application of a mass spectrometry detection method based on a single-base sequence mass label according to any one of claims 1 to 6 in the detection of low-concentration targets.

10. The use according to claim 9, characterized in that: The detection limit of the low-concentration target detection is <0.01 ng / mL.

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